Clostridial neurotoxins (tetanus and seven serotypes of botulinum neurotoxin) each block vesicular neurotransmitter release by cleaving specific proteins implicated in synaptic vesicle fusion with the presynaptic membrane. These toxins are important therapeutic agents for a number of neurologic disorders including cerebral palsy and are valuable tools for understanding neurotransmitter release, membrane trafficking, and protein sorting, transport, and targeting. We study the presynaptic action of the toxins using biochemical and morphologic techniques in primary cell cultures of fetal mouse spinal cord. Both A and E serotypes of BoNT cleave the neuronal protein SNAP-25, although BoNT A remains catalytically active within the synaptic terminal for a much longer time, suggesting differential trafficking of these toxins. Uptake of BoNT into spinal cord neurons in cell culture is enhanced greatly by potassium stimulation, implicating recycling synaptic vesicles as the uptake compartment. Movement of toxin across the vesicle membrane into the neuronal cytosol requires low intravesicular pH. Concanamycin A inhibits vacuolar-ATPase, preventing vesicle acidification. This drug traps toxin within vesicles allowing study of the synchronized translocation of toxin across the vesicle membrane. Pretreating cultures with concanamycin prevents cleavage of SNAP-25 by both BoNTs. Addition to cultures of concanamycin at times up to 15 min after toxin exposure completely abrogated BoNT/A action but did not prevent translocation of BoNT/E even when added immediately after toxin exposure. This suggests that BoNT/E trafficking proceeds beyond a pH-sensitive step more rapidly than BoNT/A. Concanamycin has no effect when added 40 min after toxin exposure, indicating that translocation of both toxins is complete by this time. This study suggests that synaptic vesicle recycling mediates uptake of BoNT/A and /E, and that these two toxin serotypes are trafficked differently after uptake. We also have monitored synaptic vesicle endocytosis and exocytosis by the quantitative kinetic measurement of synaptic staining and destaining with fluorescent FM dyes. Both BoNT A-blocked and untreated cultures are stained with FM2-10 during 5 min of potassium depolarization in the presence of dye. BoNT A treated cultures, which take up about 50% less FM dye than controls, lose only about 50% on destaining and show markedly different destaining kinetics. Current studies using electron microscopy are aimed at defining the relationship between synaptic vesicle exo- and endocytosis particularly as modified by BoNT A.